West Nile Virus is a spherical, enveloped virus containing a single-stranded positive polarity RNA genome of approximately 11 kilobases. West Nile Virus subtypes are distinguishable by antigenic variations in the envelope (E or ENV) protein and by the presence of an N-glycosylation site (Asn-Tyr-Ser) at amino acids 154-156 (Jia, 1999, Lancet. 354; 1971-2). West Nile virus is taxonomically classified within the family Flaviviridae, genus Flavivirus. The virus was originally isolated in 1937 from a febrile human who resided in the West Nile District of Uganda.
West Nile virus can be transmitted to humans and domestic animals through mosquitoes and migratory birds that serve as amplifying hosts. Although West Nile virus infection is generally asymptomatic in areas of the world where the virus is endemic, infected humans can incur a mild fever, rash, nausea, headache, disorientation and back pain. More serious complications from West Nile virus infection include hepatitis, pancreatitis, encephalitis, myocarditis, meningitis, neurologic infection, and death.
West Nile virus is geographically distributed in Africa, the Middle East, western and central Asia, India, Australia (Kunjin virus) and Europe. The first recorded epidemic occurred in Israel in the early 1950's. More recently, outbreaks of human encephalitis caused by West Nile virus have been documented in Romania and Russia. West Nile virus, introduced recently into the northeastern United States, caused seven human deaths in New York City and surrounding areas in 1999. A relatively large number of birds, particularly crows, and horses also died. The subsequent recovery of West Nile virus from mosquitoes and birds in 2000 confirmed that the virus had become established in the northeastern United States. (Anderson et al. Proc. Nat'l Acad. Sci. USA 98(23): 12885-12889, 2001).
In an attempt to prevent the spread of West Nile virus through control of larval and adult mosquitoes, mapping, spraying and removal of breeding sites has been initiated in states where West Nile virus has been identified. Despite these efforts, West Nile virus remains a threat due to its mode of transmission. At present, there is no vaccine or other known treatment for West Nile virus infection. Accordingly there remains an unmet need for reagents and methods for the diagnosis of West Nile virus infection.
During the 2002 epidemic of West Nile virus in the United States, twenty-three persons were reported to have acquired West Nile virus infection after receipt of blood components from donors infected with the virus (Morbidity and Mortality Weekly Report, 52(32): 769-772, 2003). Consequently, there is also a need for reagents and tests for diagnosing West Nile virus infection that can be used with blood or blood components such as plasma to identify infected blood.
Detection of West Nile virus using PCR-based assays has been reported. Russian patent application RU2199589, published Feb. 27, 2003, discloses a PCR-based method for detection of West Nile virus in which a double amplification assay produces a 495 base pair PCR product that is analyzed by agarose gel (from English language abstract).
WO 02/081511, published Oct. 17, 2002, assigned to Institute Pasteur and Kimron Veterinary Institute, discloses a neuroinvasive and neurovirulent strain of the West Nile virus, known as IS-98-ST1, nucleic acid molecules derived from the genome thereof and methods of detecting West Nile virus.
Anderson et al., Science 286: 2331-2333, 1999 discloses primers for amplifying a 921 base portion of the genome of West Nile virus. Lanciotti et al., J. Clin. Microbiol. 38(11): 4066-4071 discloses a PCR assay for detecting West Nile virus wherein the primers were selected from the envelope (ENV) and 3′ non-coding regions. Briese, T. et al., Emerging Infectious Diseases 8(5) May 2002 discloses a PCR assay for detecting West Nile virus wherein the primers were selected from the E gene. Huang et al. Emerging Infectious Diseases, 8(12) December 2002, discloses a PCR assay for detection of West Nile virus wherein the primers were selected from the NS5 (non-structural protein 5) and ENV genes.